Alnagar Fahima A, Brennan Paul, Brewis Ian A
Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, UK.
J Androl. 2010 Jul-Aug;31(4):393-405. doi: 10.2164/jandrol.109.008383. Epub 2010 Mar 4.
This study investigates the dynamics of serine/threonine (S/T) protein phosphorylation in sperm incubated under capacitating (C) conditions using the boar as a model system. For the first time, this approach has identified multiple dephosphorylation events that occur in a bicarbonate-dependent fashion. Different phospho-(S/T) kinase substrate antibodies were used, and dephosphorylation of 5 S/T phosphoproteins was observed in C sperm compared with noncapacitated (N) cells. Specifically, dephosphorylation of 96-, 90-, 64-, and 55-kd proteins was detected by immunoblotting using 2 phospho-Akt substrate antibodies and a phosphoprotein kinase A substrate antibody. In addition, dephosphorylation of a 105-kd protein was detected using a phospho-ATM/ATR substrate antibody. In contrast, no dephosphorylation was observed using a phosphoprotein kinase C substrate antibody, and increased tyrosine phosphorylation of 32- and 20-kd proteins was detected in C compared with N sperm. Immunolocalization experiments revealed subtle changes in the pattern expression as well as a reduction of phosphorylation in C sperm. Whereas sperm incubated in N medium containing dibutyryl cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) did not show protein dephosphorylation, incubation in C medium with dbcAMP/IBMX showed dephosphorylation as well as increased phosphorylation of other proteins (p68, p51, and p29). Finally, calyculin A, a phosphatase inhibitor, prevented dephosphorylation of p96, p90, p64, and p55 but not p105. Based on these data, we propose 2 pathways of protein dephosphorylation that are active during capacitation and independent of cAMP. Together, this provides direct evidence for more complex S/T phosphorylation dynamics than has been previously described for sperm undergoing capacitation.
本研究以公猪为模型系统，研究了在获能（C）条件下孵育的精子中丝氨酸/苏氨酸（S/T）蛋白磷酸化的动态变化。首次采用这种方法鉴定出了多个以碳酸氢盐依赖方式发生的去磷酸化事件。使用了不同的磷酸化（S/T）激酶底物抗体，与未获能（N）细胞相比，在获能精子中观察到5种S/T磷酸化蛋白发生了去磷酸化。具体而言，使用2种磷酸化Akt底物抗体和1种磷酸化蛋白激酶A底物抗体通过免疫印迹检测到96、90、64和55 kDa蛋白的去磷酸化。此外，使用磷酸化ATM/ATR底物抗体检测到105 kDa蛋白的去磷酸化。相比之下，使用磷酸化蛋白激酶C底物抗体未观察到去磷酸化，与N精子相比，在C精子中检测到32和20 kDa蛋白的酪氨酸磷酸化增加。免疫定位实验揭示了C精子中模式表达的细微变化以及磷酸化的减少。在含有二丁酰环磷腺苷（dbcAMP）和3-异丁基-1-甲基黄嘌呤（IBMX）的N培养基中孵育的精子未显示蛋白去磷酸化，而在含有dbcAMP/IBMX的C培养基中孵育则显示去磷酸化以及其他蛋白（p68、p51和p29）的磷酸化增加。最后，磷酸酶抑制剂冈田酸可阻止p96、p90、p64和p55的去磷酸化，但不能阻止p105的去磷酸化。基于这些数据，我们提出了2条在获能过程中活跃且独立于cAMP的蛋白去磷酸化途径。总之，这为精子获能过程中比先前描述的更为复杂的S/T磷酸化动态变化提供了直接证据。
